First Age and Growth Estimates in the Deep Water Shark, Etmopterus Spinax (Linnaeus, 1758), by Deep Coned Vertebral Analysis

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Mar Biol DOI 10.1007/s00227-007-0769-y

RESEARCH ARTICLE

First age and growth estimates in the deep water shark, Etmopterus Spinax (Linnaeus, 1758), by deep coned vertebral analysis

Enrico Gennari · Umberto Scacco

Received: 2 May 2007 / Accepted: 4 July 2007 © Springer-Verlag 2007

Abstract The velvet belly Etmopterus spinax (Linnaeus, by an alternation of translucent and opaque areas (Ride- 1758) is a deep water bottom-dwelling species very com- wood 1921; Urist 1961; Cailliet et al. 1983). Vertebral mon in the western Mediterranean sea. This species is a dimensions, as well as their degree of calciWcation, vary portion of the by-catch of the red shrimps and Norway lob- considerably within the elasmobranch group (La Marca sters otter trawl Wsheries on the meso and ipo-bathyal 1966; Applegate 1967; Moss 1977). For example, vertebrae grounds. A new, simple, rapid, and inexpensive vertebral of coastal and pelagic species are more calciWed than those preparation method was used on a total of 241 specimens, of bottom dwelling deep-water sharks (Cailliet et al. 1986; sampled throughout 2000. Post-cranial portions of vertebral Cailliet 1990). These diVerences are also reXected in varia- column were removed and vertebrae were prepared for age- tions of shape and in growth zone appearance, such as the ing readings. Band pair counts ranged from 0 to 9 in presence and quality of bands and/or rings. Due to these females, and from 0 to 7 in males. Von BertalanVy growth diVerences, a general protocol for the elasmobranch group equations estimated for both sexes suggested a higher is not really available because of the high variability of cal- W longevity for females (males: L1 = 394.3 mm k =0.19 ci cation degree among species (Applegate 1967; Cailliet W t0 = ¡1.41 L0 = 92.7 mm A99 = 18.24 years; females: L1 = et al. 1983). Based on identi cation and count of band 450 mm k =0.16 t0 = ¡1.09 L0 = 72 mm A99 = 21.66 years). pairs, many techniques have been developed to assess age Age estimations are discussed in the context of deep water in cartilaginous species, assuming an annual periodicity shark species. This is the Wrst successful attempt at delin- (Cailliet et al. 1990). Even if deposition time should be eating faint growth bands in the poorly calciWed deep coned properly validated within each species (Beamish and vertebrae of E. spinax. This technique may be used in other McFarlane 1983; Cailliet 1990; Campana 2001), age esti- diYcult poorly calciWed species. mates have not always been validated (Campana 2001). Etmopterus spinax is a bottom dwelling shark, typical of the bathyal stratum (200–1,000 m depth) of the Mediterra- Introduction nean sea (Tortonese 1956; Fischer et al. 1987; Notarbartolo Di Sciara and Bianchi 1998). It is caught as by-catch by In cartilaginous Wsh, hard and calciWed structures, such as commercial otter trawlers (Bertrand et al. 2000; Relini et al. spines and vertebral centra, exhibit growth zones, identiWable 2000). E. spinax is characterized, as well as other deep bottom dwelling sharks (Etmopterus baxteri, Galeus melastomus, Galeorhinus galeus), by typical amphicoelous “deep coned” vertebrae; additionally its vertebrae are very small (Fig. 1), have little calciWcation and outer margins tend to refold on themselves as Wsh size increases (Fig. 11). There- Communicated by R. Cattaneo-Vietti. fore, these features, together with the faint markings and fragile condition of the vertebrae, render them unsuitable to E. Gennari · U. Scacco (&) I.C.R.A.M, Via di Casalotti 300, 00166 Rome, Italy both most traditional ageing and validating increment peri- e-mail: [email protected] odicity techniques. The simple, inexpensive, and rapid 123 Mar Biol

Fig. 2 Trawl grounds oV the Latium coast where Etmopterus spinax specimens were sampled

Fig. 1 Typical vertebral size compared to an observer’s Wnger. Scale bar 1 mm water, it was dipped again in a new but same concen- trated NaClO–water solution, for the same amount of method proposed in this work, modiWed from Hoenig and time, and so on until complete removal of connective Brown (1988), produced band elucidation for E. spinax ver- and muscular tissues, and separation of each single vertebrae, leading to the Wrst, but unvalidated, age results for tebra. this species. 3. To measure the vertebral length (VL), each vertebra was put under transmitted light on a stereo-microscope provided with digital still camera, and millimeter paper Materials and methods for calibration. A digital image was acquired, and mea- surements were taken using Image-Pro©Plus. Due to Samples were obtained from commercial otter trawl opera- the small sizes of these vertebrae, VL, rather than tions, conducted by Fiumicino marine, oV the Latium coast vertebral diameter, was chosen to analyze vertebral (central Tyrrhenian sea) (Fig. 2). A combined eVort of 16 growth. hauls distributed in 3 bathymetrical strata (300–490 m, 4. Each centrum was dipped into a 5% in volume W 491–580 m, and 581–900 m) was made seasonally during Co(NO3)2–H2O solution in order to stain calci ed 2000. A total of 241 specimens of Etmopterus spinax areas. Time of immersion varied between 1 and 5 min, (Linnaeus 1758) were collected and dissected. Each speci- depending on vertebral dimension and hence on its men was sexed (152 females and 89 males), measured (over degree of calciWcation. Each test tube had to be stirred the body total length and pre-caudal length, TL and PL gently in order to allow Co(NO3)2penetration into the respectively) to the nearest millimetre, and weighed (evis- pronounced cavities of the deep-coned vertebra. cerated weight, WE) to the nearest centigram. According to 5. Vertebral centrum was rinsed with distilled water for the estimates of velvet belly size at Wrst maturity in the few seconds in order to remove excess Co(NO3)2. Mediteranean sea (Vacchi and Relini Orsi 1979; Fischer 6. To enhance the bands, each centrum was dipped into an et al. 1987), specimens were divided into 3 size classes: alcohol acid–water solution (obtained adding hydro- “juveniles” (I: 100–200 mm TL, n = 56), “subadult” (II: chloric acid into a 70% in volume ethanol–water solu- 201–301 mm TL, n = 139), and “adult” (III: 301–435 mm tion, with a ratio of 1:20) between few seconds to one TL, n = 46). Sex ratio was calculated for size class as fol- minute, depending on vertebral dimension. Time of low: Females/(Females + Males) 100. A small post-cranial immersion was the most critical step of the entire prep- section of vertebral column (3–4 vertebrae) was removed aration: a shorter time could not allow band enhance- from each specimen. ment, whereas a prolonged dipping could destroy the Vertebrae were prepared according to the method pro- centrum. posed by Hoenig and Brown (1988), but modiWed for this 7. After being rinsed using distilled water, the stained study as follow: centrum was then viewed under the transmitted light using a stereo-microscope, and a digital image was 1. After removal, vertebral column section was stored at obtained and read independently by two readers. ¡20°C. 2. Section was dipped in a 10% in volume NaClO–water The criterion chosen for band readings is based on the iden- solution for 15 min. After being rinsed using distilled tiWcation of a dark layer followed by a lighter one deWned 123 Mar Biol as a band pair (Cailliet et al. 2006). Each band was consid- DiVerence in percent of occurrence between type errors ered a temporal growth zone (Fig. 3). (§1 and §2) was tested by a Chi-square test. Each reader counted, twice for each centrum, dark bands Estimates of precision were evaluated following the on the vertebral outer surface, from the core toward the dis- methodology suggested by Goldman (2004): total percent- tal margin (EA = estimated age). Counting was performed age agreement (PA), percentage agreement plus-minus one by each reader without knowledge either of the specimen band (PA § 1) and percentage agreement by size classes length or the other reader’s count. PA (I, II, III) were calculated both between and within readers. Chi-square test on contingency tables was utilized Statistical analysis to check for bias in all these cases. The age-bias curve (Campana et al. 1995) was utilized to test for bias between DiVerences in sex ratio among sizes classes were tested by readers within age groups. In addition the non-parametric a chi-square Test on a 2 £ 3 contingency table. Wilcoxon Test (Conover 1971), index of average percent Linear regression analysis was used to calculate length– error (IAPE), and mean coeYcient of variation (CV) weight relationships (total length on transformed natural (Beamish and Fournier 1981) were calculated in order to logarithm of eviscerated weight), as well as the VL-TL, and provide further estimates of precision in band count estima- EA-TL correlations. A natural logarithmic transformation tion between readers. The statistical package Statistica (6.0) of (EA + 1) was also needed due to the presence of “0” val- 2006 was used for the aforementioned analyses. ues. ANCOVA analysis was then used to investigate sex The von BertalanVy growth parameters were determined diVerences for all the aforementioned relationships. for each sex using the Wshery program FISHPARM (Prager ¡k(t ¡ t0) et al. 1987), using the equation L(t)=L1(1 ¡ e ) (BertalanVy 1960). Longevity was estimated through the

algorithm A99 = 5·Ln(2)/k (Fabens 1965), where A99 is the time (in years) passed before reaching 99% of L1 and k is the growth constant derived from the von BertalanVy

growth equation. Extrapolated longevity, based upon A99, is believed to give a more realistic estimate of the maximum

age rather than A97 and A95 (Skomal and Natanson 2003).

Results

Systematic error did not occurr in estimates of PA, PA § 1, and PA by length classes both between readers and within readers 1 and 2 (Table 1). PA by size classes decreased sig- niWcantly with increasing Wsh length both between and within readers, resulting the lowest in the largest size class. The conWdence intervals of all age groups (Fig. 4) enclosed the reference diagonal (line that bisects graph area passing through the origin with an angle of 45°) demonstrating no signiWcant bias occurred in band counting within all age groups between readers. Error type § 1 band occurred at frequency (14.11%) signiWcantly (2 =14.22, df =1, P < 0.001) much higher than type § 2 bands (0.83%) between readers whose counts did not show signiWcant diVerence (Wilcoxon Test, T = 297.50, n =241, P <0.6; IAPE = 1.86%, mean CV = 1.02%). Ten (0+ to 9) and eight (0+ to 7) age groups were found in females and males, respectively (Fig. 5). Band deposition periodicity was assumed annual due to the impossibil- Fig. 3 MagniWed digital images of deep coned vertebrae of Etmopte- ity of validating it. rus spinax immersed in rinsed water and observed through stereomi- W 2 croscope transmitted light after band enhancing treatment. Upper one Females signi cantly ( = 16.71, df =2, P < 0.001) band (age group 1), male, 150 mm TL; Lower Wve bands (age group 5), outnumbered males in all sizes classes with the most dramatic female, 275 mm TL. Scale bar 1 mm diVerence in the adult class (I = 58.93%, II = 56.12%, 123 Mar Biol

Table 1 Precision in estimate of total percent agreement (PA), percent agreement plus/minus one band (PA § 1) and percent agreement by size classes (PA I, PA II, PA III) between and within readers in Etmopterus spinax from central Tyrrhenian Sea Between readers Within reader 1 Within reader 2

PA 85.06% 89.21% 85.48% df 111 2 59.25 74.11 60.66 P <0.001 <0.001 <0.001 PA § 1 99.17% 98.34% 97.51% df 111 2 116.53 112.63 108.80 P <0.001 <0.001 <0.001 PA (I) 87.50% 92.86% 87.50% PA (II) 88.49% 91.37% 89.93% Fig. 4 Age bias graph for age groups of Etmopterus spinax. Dotted PA (III) 71.74% 78.26% 69.56% line, vertical bars and numbers above refer respectively to main diag- df 222 onal, mean estimated age conWdence intervals and sample size for each age groups 2 7.97 7.17 9.40 P <0.05 <0.05 <0.01

higher slope for the female regression therefore indicating III = 89.13%). Since no diVerences were found in the that estimated age varied with vertebral length depending slopes of TL-EW and of TL-VL relationships between on sex. For this reason the Von BertalanVy growth equation sexes, data were pooled resulting in single regressions was calculated separately for females (Fig. 9) and males (Figs. 6, 7, respectively). Mean eviscerated weight and ver- (Fig. 10). Males attained a smaller L1 with a higher k value tebral length resulted both higher in females compared to than females (Table 2). Estimated size at birth (L0) and lon- males (ANCOVA, F1,238 = 5.02, P < 0.05 and F1,238 =6.45, gevity (A99) were 72.0 mm and 21.66 years, respectively, P < 0.05, respectively). Conversely, VL-EA relationships for females, and 92.7 mm and 18.24 years, respectively, for (Fig. 8) diVered between sexes, revealing a signiWcantly males.

(a) (b)

Fig. 5 Age groups of females (white bars) and males (dark bars) of Etmopterus spinax in the four seasons (a winter, b spring, c summer, d autumn) from central Tyrrhenian Sea 123 Mar Biol

Fig. 6 Relationship between total length (TL) and eviscerated weight Fig. 8 Relationships (Test for Homogeneity of Slope: F1,237 = 10.25, 2 (EW) (F1,239 = 15,672.59, R =0.98, P < 0.001) for pooled data (Test P < 0.01) between vertebral length (VL) and transformed natural log- W for Homogeneity of Slope: F1,237 = 2.65, P < 0.10) of males (open arithmic of estimated age (EA) in males ( lled circle) (F1,87 = 240.76, W 2 2 circle) and females ( lled square) of Etmopterus spinax from central R = 0.73, P < 0.001) and females (diamond) (F1,150 = 469.10 R =0.76, Tyrrhenian Sea P < 0.001) of Etmopterus spinax from central Tyrrhenian Sea

Fig. 7 Relationship between total length (TL) and vertebral length 2 Fig. 9 Von BertalanVy growth curve in Etmopterus spinax males (VL) (F1,239 = 4,201.8, R = 0.94, P < 0.001) for pooled data (Test for from central Tyrrhenian Sea Homogeneity of Slope: F1,237 = 0.33, P <0.6) of males (asterisk) and females (diamond) of Etmopterus spinax from central Tyrrhenian Sea

E. princeps, and for Squalus blainvillei, respectively. All these similar observations conWrmed the hypothesis of the Discussion latter authors that growth rate is similar between sexes in elasmobranchs untill sexual maturation, then rate starts to The proportion of females found in this study increased in decrease independently by sex. In fact according to Vacchi the bigger/older size class, as observed in other Mediterra- and Relini Orsi (1979), E. spinax males mature earlier than nean areas (Punnett 1904; Vacchi and Relini Orsi 1979; females (280 vs. 340 mm TL) in the Gulf of Genoa. Sion et al. 2002; Cecchi et al. 2004). The validity of the adopted method was ascertained by The dimorphism in size found between sexes is con- the very low level of disagreement found in band count Wrmed both by a higher mean weight and a bigger mean within and between observers and within each age group, vertebral size for females. Nevertheless, the ontogeny of according to combined methodology suggested by Calliet both variables was similar between sexes. Cecchi et al. et al. (2006). Also a CV of less than 7.6%, corresponding to (2004) described a comparable trend in mean weight for the an IAPE of 5.5%, is considered to be adequate for age esti- same species, as Jakobsdóttir (2001) and Cannizzaro et al. mation precision obtained by band counts in vertebral (1995) previously reported for the Atlantic co-generic structures (Campana 2001). The low level of disagreement 123 Mar Biol

Fig. 10 Von BertalanVy growth curve in Etmopterus spinax females from central Tyrrhenian Sea Fig. 11 Example of refolding of larger specimens’ vertebrae. Scale bar 1 mm

Table 2 Asymptotic standard error (ASE), CoeYcient of Variation Vertebral folding is the main reason why increment peri- L k (CV) and estimate for asymptotic length ( 1), rate of growth ( ), time odicity was not conWrmed in this study, since validating (t0) and length (L0) at birth in males and females of Etmopterus spinax from central Tyrrhenian Sea increment periodicity is highly recommended across the entire age range (Beamish and McFarlane 1983; Campana Parameter Females Males 2001). Even in the co-generic E. baxteri, validation was not Estimate ASE CV Estimate ASE CV possible since most traditional techniques are unsuitable for deep water species (Irvine et al. 2006). L (mm) 450 46.33 0.01 394.3 59.72 0.15 1 Notwithstanding problems in working with so little, K (years¡1) 0.16 0.04 0.24 0.19 0.07 0.38 fragile and poorly calciWed vertebrae, the new methodology t (years) ¡1.09 0.39 ¡0.36 ¡1.41 0.59 ¡0.42 0 proposed in this study was able to successfully enhance L (mm) 72 92.7 0 vertebral bands. In fact, both calculated asymptotic length and size at birth in this study agree with the data reported for E. spinax in the Mediterranean Sea (Fischer et al. 1987), observed in this study was probably due to the high deWni- even though size at birth was slightly underestimated for tion digital images utilized for counting. Nevertheless, females. Those authors indicated that the maximum disagreement between observers increased as Wsh size reported size is 450 mm TL, and that newborns range increased, and the most common error was plus/minus one between 90–110 mm in size, although some other authors band as also Correia and Figueiredo (1997) reported for the stated the occurrence of 85 mm TL newborns in the Gulf of deep water blackmouth catshark Galeus melastomus. Genoa (Vacchi and Relini Orsi 1979). Misjudgements are usually attributed to many reasons Growth parameters and age groups found in this study (Campana 2001; Skomal and Natanson 2003): Wrstly, ran- are similar to those calculated, through spine age analysis dom error probability among readers could increase (Sion et al. 2002) and indirect methods (i.e Bhattacharya, because of a greater number of bands to count on larger Normsep) (Cecchi et al. 2004), from Ionian and northern specimens, since they have more bands than smaller co- Tyrrhenian Sea populations of E. spinax, respectively. speciWcs. A further source of bias might be originated from From the Wrst study, only 7 age classes were found, rather the weak separation between distal bands, which is due to than 9 found in this study. From the second study, both L1 reduced growth rate in older individuals (BertalanVy 1960; and k were a little diVerent compared to this study, resulting Cortés 2000), and therefore bands tend to be deposited at in a slight higher maximum estimated age for both males smaller intervals as vertebral length increases. Moreover, and females. Such diVerences may be due either to method- such disagreement might be explained since some distal ology, or to real diVerences in comparing natural popula- bands remained hidden to observers because in some tions (Tanaka et al. 1990; Skomal and Natanson 2003). sharks, for instance E. spinax, outer vertebral margins tend Another important conWrmation of the validity of this to refold on themselves in older specimens as vertebral size study, is given by the fact that males result to grow faster increases (Fig. 11); therefore preventing reading and mea- and reach a smaller L1 compared to females, which conse- surements of the latest rings in the older specimens. quently grow older (10 vs. 8 age classes for females and 123 Mar Biol males, respectively). The higher longevity and size in a more favourable environment, could play an impor- achieved by females is a common feature in several species tant role in aVecting the growth rate of deep water sharks of elasmobranchs (Cortés 2000): it was ascertained in such as E. spinax, which is well adapted to these ecosys- coastal (Ferreira and Vooren 1991) and deep-water squali- tems. formes (Rey et al. 2002), in large pelagic species (Parker Extrapolated longevity shows that, despite its size, E. and Stott 1965; Cailliet et al. 1985) and squatiniformes spinax is a long-living animal, with females attaining (Natanson 1984). Therefore, since elasmobranch males greater age than males, as it is typical of most elasmo- attain size at Wrst maturity earlier than females (then, their branchs (Cortés 2000). These features, together with late growth rate sharply decreases), diVerences in maximum maturity and low fecundity, render such a taxon greatly length and longevity between sexes are due to a delayed vulnerable to over Wshing (Stevens et al. 2000), both as tar- female maturation. In this way, the occurrence of bigger geted and by-catch commercial species. females in mature populations deals with an evolutionary In conclusion, this new technique is able to delineate advantage because of the greater fecundity of bigger speci- faint growth bands in the poorly calciWed and fragile deep mens (Cortés 2000). coned vertebrae of E. spinax. Therefore this methodology Comparing E. spinax growth rate with data available for may be used in the future, in order to successfully outline similar-size Mediterranean squaliformes, it grows with a vertebral increments in other diYcult species. rate more similar to the coastal S. blainvillei (Cannizzaro et al. 1995) than to the deep water catshark G. melastomus Acknowledgments We would like to thank for their support and (Correia et al. 1999 for Atlantic waters). In fact, growth rate help: M. Vacchi, G.D. Ardizzone, G. La Mesa, M. La Mesa, J. Bruner of E. spinax is unusually high within deep water species, and C. Gubili. We are grateful to L. Natanson for many constructive if compared, for instance, to Centrophorus squamosus comments and help, which greatly improved the manuscript. (Clarke et al. 2002) and Squalus mitsukurii (Taniuchi and Tachikawa 1999) for other areas. 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